Rat cauda epididymidal spermatozoa are kept quiescent in the cauda lumen by a protein fraction termed "immobilin", but it has not been shown whether or not this activity is unique to the immobilin fraction. Cauda fluid was subjected to gel filtration chromatography and the eluents contributing to three peaks of absorbance at A280 were pooled, and reconstituted at equivalent concentrations. Their relative viscoelasticity and their effects on sperm motility were determined. Peak 1 (P1), containing proteins greater than 400 kd, retained the greatest sperm-immobilizing activity but P2 and P3 also had sperm-immobilizing activity related to their viscoelasticity. P1 and an immobilin fraction obtained by ultracentrifugation of rat cauda fluid were generally similar in their sperm-immobilizing activity, viscoelasticity index, electrophoretic patterns, and binding characteristics to Concanavalin A; perm-immobilizing factor obtained by gel filtration chromatography and the immobilin fraction obtained by centrifugation are believed to be the same products. Further, it was shown that intraluminal intraluminal testicular fluids did not inhibit cauda sperm motility even at epididymal-like protein concentrations; thus, it is believed that the sperm-immobilizing factor(s) is of epididymal, not testicular, origin.